JP2009120486A - New activator of orphan receptor in nucleus and application of the same - Google Patents

New activator of orphan receptor in nucleus and application of the same Download PDF

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JP2009120486A
JP2009120486A JP2005350440A JP2005350440A JP2009120486A JP 2009120486 A JP2009120486 A JP 2009120486A JP 2005350440 A JP2005350440 A JP 2005350440A JP 2005350440 A JP2005350440 A JP 2005350440A JP 2009120486 A JP2009120486 A JP 2009120486A
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pregnane
therapeutic agent
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Toshiyuki Shimizu
俊之 清水
Takuro Niwa
卓朗 丹羽
Hiroshi Chiba
寛 千葉
Masakiyo Hosokawa
正清 細川
Kaoru Kobayashi
かおる 小林
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Chiba University NUC
Mitsubishi Pharma Corp
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Mitsubishi Pharma Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C07D237/34—Phthalazines with nitrogen atoms directly attached to carbon atoms of the nitrogen-containing ring, e.g. hydrazine radicals
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new activator of an orphan receptor in a nucleus, useful as a therapeutic agent of liver diseases, metabolic disorders, etc. <P>SOLUTION: This compound expressed by general formula (I) [R<SP>1</SP>is cyclohexyl, a phenyl having ≥1 substituent, a thienyl having ≥1 substituent, a furyl having ≥1 substituent, thiazolyl, phenoxy, a 7-9C phenylalkyl; phenylthio, morphorino, piperidyl, pyrrolidinyl, pyridyl or imidazolyl; R<SP>2</SP>is -CHR<SP>3</SP>R<SP>4</SP>or cyclohexyl; and ring A is a benzene ring, thiophene ring or furan ring], its pharmaceutically acceptable salt or its solvated material is used as a pregnane receptor activator. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、核内オーファン受容体の新規活性化剤及びその用途に関する。より詳しくは、肝疾患や代謝性疾患の治療薬などとして有用なプレグナンＸ受容体（以下、ＰＸＲと呼ぶ）活性化剤及びそれを含有する医薬に関する。   The present invention relates to a novel activator of a nuclear orphan receptor and use thereof. More specifically, the present invention relates to a pregnane X receptor (hereinafter referred to as PXR) activator useful as a therapeutic agent for liver diseases and metabolic diseases, and a medicine containing the same.

ＰＸＲはリガンドが結合すると標的遺伝子の転写調節配列に結合して標的遺伝子の転写を活性化する核内受容体の一つである。その生体内リガンドが明らかにされていないことからオーファン受容体に分類されるが、リファンピシンやリトコール酸などがＰＸＲを介してＣＹＰ（チトクロームＰ４５０）３Ａ４などの標的遺伝子の発現を増加させることが知られている（非特許文献１）。
ＣＹＰ３Ａ４は肝臓や小腸で恒常的に発現しており、コレステロールの代謝産物であるリトコール酸や抗結核薬のリファンピシンなどで誘導されることが知られている。また、ＣＹＰ３Ａ４は様々な生体異物のみならず、生体内の胆汁酸、リトコール酸やデオキシコール酸を酸化し、***させる作用を有する。ＣＹＰ３Ａ４の誘導剤であるリファンピシンを胆汁うっ滞性の肝疾患患者に投与することによって生体内の余分なビヒルビン量が減少し、治療効果が得られているという報告や、リファンピシンが初期の胆汁性肝硬変の際に認められる深刻なそう痒治療にも効果があるという報告などがある（非特許文献２）。これらのことから、ＰＸＲを介してＣＹＰ３Ａ４などの標的遺伝子を活性化することができればこれらの疾患の治療剤として有用であると考えられる。
しかしながら、ＰＸＲのリガンドはリファンピシンやリトコール酸などの化合物以外にはあまり知られていなかった。
一方、フタラジン誘導体について種々の薬理作用が報告されているが（特許文献１〜４）、フタラジン誘導体がＰＸＲを活性化することや、ＣＹＰ３Ａ４を誘導することは知られていなかった。
特開平６０−２４３０７４号公報 特開平６−１３５９３８号公報 特開平８−３４７３４号公報 特開平１０−１０９９３６号公報 Gastroenterology. 2005 Aug;129(2):735-40. Gastroenterology. 2005 Aug;129(2):476-85. PXR is one of nuclear receptors that binds to a transcriptional regulatory sequence of a target gene and activates transcription of the target gene when a ligand is bound. Although its in vivo ligand has not been clarified, it is classified as an orphan receptor, but it is known that rifampicin, lithocholic acid, etc. increase the expression of target genes such as CYP (cytochrome P450) 3A4 via PXR. (Non-Patent Document 1).
CYP3A4 is constantly expressed in the liver and small intestine, and is known to be induced by lithocholic acid, which is a metabolite of cholesterol, rifampicin, which is an antituberculosis drug, and the like. CYP3A4 has an action of oxidizing and excreting not only various xenobiotics but also bile acid, lithocholic acid and deoxycholic acid in the living body. There are reports that the administration of rifampicin, an inducer of CYP3A4, to patients with cholestatic liver disease has reduced the amount of extra bihilbin in the body, and a therapeutic effect has been obtained, and rifampicin is an early biliary cirrhosis There is a report that it is also effective for the treatment of severe pruritus recognized at the time of treatment (Non-patent Document 2). From these facts, if a target gene such as CYP3A4 can be activated via PXR, it is considered useful as a therapeutic agent for these diseases.
However, PXR ligands are not well known except for compounds such as rifampicin and lithocholic acid.
On the other hand, although various pharmacological actions have been reported for phthalazine derivatives (Patent Documents 1 to 4), it has not been known that phthalazine derivatives activate PXR or induce CYP3A4.
JP-A-60-243074 Japanese Patent Laid-Open No. 6-135938 JP-A-8-34734 JP-A-10-109936 Gastroenterology. 2005 Aug; 129 (2): 735-40. Gastroenterology. 2005 Aug; 129 (2): 476-85.

本発明は肝疾患や代謝性疾患の治療薬などとして有用なＰＸＲ活性化剤を提供することを課題とする。   An object of the present invention is to provide a PXR activator useful as a therapeutic agent for liver diseases and metabolic diseases.

本発明者は上記課題を解決すべく、鋭意検討を行った。その結果、下記一般式（Ｉ）で示される化合物がＰＸＲを活性化することを見出し、本発明を完成するに至った。   The present inventor has intensively studied to solve the above problems. As a result, it was found that a compound represented by the following general formula (I) activates PXR, and the present invention was completed.

すなわち、本発明は以下のとおりである。
（１）下記一般式（I）で表される化合物もしくはその医薬的に許容される塩、又はそれらの溶媒和物を含むプレグナンＸ受容体活性化剤。

Figure 2009120486
上記式中で、Ｒ1 はシクロヘキシル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいチエニル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフリル基；チアゾリル基；フェノキシ基；Ｃ7〜Ｃ9のフェニルアルキル基；フェニルチオ基；モルフォリノ基；ピペリジル基；ピロリジニル基；ピリジル基；又はイミダゾリル基を表し、
Ｒ2 は−ＣＨＲ3 Ｒ4 （Ｒ3 は水素原子またはＣ1 〜Ｃ4 のアルキル基を表し、Ｒ4 はＣ1 〜Ｃ4 のアルキル基；シクロヘキシル基；チエニル基；またはＣ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基を表す。）；又はシクロヘキシル基を表し、
環Ａはベンゼン環、チオフェン環またはフラン環を表す。
（２）（１）のプレグナンＸ受容体活性化剤を含むＣＹＰ３Ａ４誘導剤。
（３）（１）のプレグナンＸ受容体活性化剤を含む肝疾患治療薬。
（４）肝疾患が胆汁うっ滞性肝疾患又は黄疸である、（３）の肝疾患治療薬。
（５）（１）のプレグナンＸ受容体活性化剤を含む急性腎疾患治療薬。
（６）（１）のプレグナンＸ受容体活性化剤を含む代謝性疾患治療薬。 That is, the present invention is as follows.
(1) A pregnane X receptor activator comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof.
Figure 2009120486
 In the above formula, R 1 represents a cyclohexyl group; C 1 -C 4 alkyl groups, C 1 -C 4 one or more may have a substituent phenyl group selected from an alkoxy group and a halogen atom; C 1 -C 4 alkyl groups, C 1 -C 4 one or more thienyl group which may have a substituent selected from alkoxy groups and halogen atoms, alkyl groups of C 1 ~C 4, C 1 ~C A furyl group optionally having one or more substituents selected from 4 alkoxy groups and a halogen atom; a thiazolyl group; a phenoxy group; a C 7 to C 9 phenyl alkyl group; a phenylthio group; a morpholino group; a piperidyl group; Represents a pyrrolidinyl group; a pyridyl group; or an imidazolyl group,
R 2 represents —CHR 3 R 4 (R 3 represents a hydrogen atom or a C 1 to C 4 alkyl group, R 4 represents a C 1 to C 4 alkyl group; a cyclohexyl group; a thienyl group; or C 1 to C 4. alkyl group, optionally having one or more substituents selected from alkoxy groups and halogen atoms of C 1 -C 4 represents a phenyl group);. or cyclohexyl group,
Ring A represents a benzene ring, a thiophene ring or a furan ring.
(2) A CYP3A4 inducer comprising the pregnane X receptor activator according to (1).
(3) A therapeutic agent for liver disease comprising the pregnane X receptor activator according to (1).
(4) The therapeutic agent for liver disease according to (3), wherein the liver disease is cholestatic liver disease or jaundice.
(5) A therapeutic agent for acute kidney disease comprising the pregnane X receptor activator according to (1).
(6) A therapeutic agent for metabolic diseases comprising the pregnane X receptor activator according to (1).

本発明のＰＸＲ活性化剤は、胆汁うっ滞性肝疾患や黄疸などの肝疾患、高コレステロール血症などの代謝性疾患など、ＰＸＲを活性化することによって治療しうる疾患の治療に有効に使用することができる。   The PXR activator of the present invention is effectively used for treatment of diseases that can be treated by activating PXR, such as cholestatic liver diseases, liver diseases such as jaundice, and metabolic diseases such as hypercholesterolemia. can do.

本発明のＰＸＲ活性化剤は、一般式（I）の化合物もしくはその医薬的に許容される塩、又はそれらの溶媒和物を有効成分として含む。

Figure 2009120486
ここで、Ｒ1 はシクロヘキシル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基；Ｃ1
〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいチエニル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフリル基；チアゾリル基；フェノキシ基；Ｃ7〜Ｃ9のフェニルアルキル基；フェニルチオ基；モルフォリノ基；ピペリジル基；ピロリジニル基；ピリジル基；又はイミダゾリル基を表し、
Ｒ2 は−ＣＨＲ3 Ｒ4 （Ｒ3 は水素原子又はＣ1 〜Ｃ4 のアルキル基を表し、Ｒ4 はＣ1 〜Ｃ4 のアルキル基；シクロヘキシル基；チエニル基；またはＣ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基を表す。）；又はシクロヘキシル基を表し、
環Ａはベンゼン環、チオフェン環又はフラン環を表す。 The PXR activator of the present invention contains a compound of general formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.
Figure 2009120486
 Wherein, R 1 represents a cyclohexyl group; C 1 -C 4 alkyl groups, C 1 -C 4 one or more may have a substituent phenyl group selected from an alkoxy group and a halogen atom; C 1
-C 4 alkyl groups, C 1 -C 4 one or more may have a substituent group thienyl group selected from an alkoxy group and a halogen atom; C 1 -C 4 alkyl groups, C 1 -C 4 1 or more may have a substituent group furyl group selected from alkoxy groups and halogen atoms; phenylalkyl C 7 -C 9;; a thiazolyl group; a phenoxy group, phenylthio group; a morpholino group; piperidyl group; pyrrolidinyl A group; a pyridyl group; or an imidazolyl group,
R 2 represents —CHR 3 R 4 (R 3 represents a hydrogen atom or a C 1 to C 4 alkyl group, R 4 represents a C 1 to C 4 alkyl group; a cyclohexyl group; a thienyl group; or C 1 to C 4. alkyl group, optionally having one or more substituents selected from alkoxy groups and halogen atoms of C 1 -C 4 represents a phenyl group);. or cyclohexyl group,
Ring A represents a benzene ring, a thiophene ring or a furan ring.

上記一般式中のＣ1 〜Ｃ4 のアルキル基としてはメチル基、エチル基、ｎ−プロピル基、ｉ−プロピル基、ｎ−ブチル基、ｔ−ブチル基等が挙げられ、Ｃ1 〜Ｃ4 のアルコキシ基としてはメトキシ基、エトキシ基、ｎ−プロポキシ基、ｉ−プロポキシ基、ｎ−ブトキシ基、ｔ−ブトキシ基等が挙げられ、ハロゲン原子としてはフッ素原子、塩素原子、臭素原子等が挙げられる。
Ｒ1 としてはフェニル基、２−チエニル基または２−フリル基が好ましく、特にフェニル基が好ましい。Ｒ2 としては−ＣＨＲ3 ′Ｒ4 ′（Ｒ3 ′はＣ1 〜Ｃ4 のアルキル基を表し、Ｒ4 ′はシクロヘキシル基を表す。）が好ましく、特に

Figure 2009120486
が好ましい。環Ａとしてはベンゼン環またはチオフェン環を表す化合物が好ましく、特にベンゼン環が好ましい。 C 1 -C 4 methyl group of an alkyl group in the general formula, ethyl group, n- propyl group, i- propyl, n- butyl, t- butyl group and the like, C 1 -C 4 Examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, and a t-butoxy group, and examples of the halogen atom include a fluorine atom, a chlorine atom, and a bromine atom. It is done.
R 1 is preferably a phenyl group, a 2-thienyl group or a 2-furyl group, and particularly preferably a phenyl group. R 2 is preferably —CHR 3 ′ R 4 ′ (R 3 ′ represents a C 1 -C 4 alkyl group, and R 4 ′ represents a cyclohexyl group), particularly.
Figure 2009120486
 Is preferred. As ring A, a compound representing a benzene ring or a thiophene ring is preferable, and a benzene ring is particularly preferable.

PXR活性化剤の有効成分として用いることのできる一般式（Ｉ）の化合物の好ましい例として、具体的には、下記式で表される化合物が挙げられる。この化合物を後述の実施例では化合物１と呼ぶ。

Figure 2009120486
その他に実施例で例示したような化合物も、PXR活性化剤の有効成分として用いることができる。 Preferable examples of the compound of the general formula (I) that can be used as an active ingredient of the PXR activator include specifically the compounds represented by the following formula. This compound is referred to as Compound 1 in the examples described below.
Figure 2009120486
 In addition, compounds as exemplified in the examples can also be used as active ingredients of the PXR activator.

上記一般式（I）で表される化合物は公知の化合物であり、その製造方法は特に制限されないが、例えば、特開平６−１３５９３８号公報、特開平８−３４７３４号公報、特開平１０−１０９９３６号公報などに記載された方法に従って製造することができる。   The compound represented by the above general formula (I) is a known compound, and its production method is not particularly limited. For example, JP-A-6-13538, JP-A-8-34734, JP-A-10-109936 It can be produced according to the method described in Japanese Patent Publication.

一般式（Ｉ）のアミノピリダジン誘導体の医薬的に許容しうる塩としては、酸付加塩又は塩基付加塩を用いることができるが、生理的に許容されるものであれば塩の種類は特に限定されることはない。
一般式（Ｉ）の化合物およびその塩は溶媒和物（水和物も含む）の形で存在することもあるので、これらの溶媒和物（水和物も含む）も本発明のPXR活性化剤の有効成分として
用いることができる。
さらに上記一般式（Ｉ）のアミノピリダジン誘導体に不斉炭素が存在する場合は、（Ｒ）体、（Ｓ）体、（ＲＳ）体のいずれをもとることができ、これらはすべて本発明のPXR活性化剤の有効成分として用いることができる。 As the pharmaceutically acceptable salt of the aminopyridazine derivative of the general formula (I), an acid addition salt or a base addition salt can be used, and the kind of the salt is particularly limited as long as it is physiologically acceptable. It will never be done.
Since the compounds of the general formula (I) and salts thereof may exist in the form of solvates (including hydrates), these solvates (including hydrates) also activate the PXR of the present invention. It can be used as an active ingredient of an agent.
Furthermore, when an asymmetric carbon exists in the aminopyridazine derivative of the above general formula (I), any of (R), (S), and (RS) can be taken, and these are all of the present invention. It can be used as an active ingredient of a PXR activator.

一般式（Ｉ）の化合物もしくはその医薬として許容される塩又はそれらの溶媒和物を、そのまま、あるいは薬学的及び製剤学的に許容される担体と組み合わせることにより、ＣＹＰ３Ａ４誘導剤、さらには、ＣＹＰ３Ａ４の誘導及び／又はその他のＰＸＲ標的遺伝子の誘導を介して治療しうる疾患の治療剤として使用することができる。
本発明のＰＸＲ活性化剤を用いて治療しうる疾患としては、うっ滞性肝疾患や黄疸などの肝疾患、急性腎疾患などを挙げることができる。
ＰＸＲの活性化とこれらの疾患の治療効果との関係については、例えば、以下のような文献に記載されている。
うっ滞性肝疾患 特表2003-535912号公報 Proc Natl Acad Sci U S A. 2001;98(6):3369-74.
黄疸 Hepatology. 2005; 41(3):497-505.
急性腎疾患 Proc Natl Acad Sci U S A. 2005;102(6):2198-203
さらに、ＰＸＲを活性化するとコレステロールの分解が促進するため、本発明のＰＸＲ活性化剤は、高血圧、動脈硬化、高脂血症、肥満、高コレステロール血症などの代謝性疾患の治療薬としても有用である（米国特許出願公開2004/0019027号明細書）。 By combining the compound of general formula (I) or a pharmaceutically acceptable salt thereof or a solvate thereof as it is or with a pharmaceutically and pharmaceutically acceptable carrier, a CYP3A4 inducer, further, CYP3A4 It can be used as a therapeutic agent for diseases that can be treated through induction of and / or induction of other PXR target genes.
Examples of the diseases that can be treated using the PXR activator of the present invention include stasis liver diseases, liver diseases such as jaundice, and acute kidney diseases.
The relationship between the activation of PXR and the therapeutic effect of these diseases is described, for example, in the following documents.
Stalling Liver Disease Special Table 2003-535912 Publication Proc Natl Acad Sci US A. 2001; 98 (6): 3369-74.
Jaundice Hepatology. 2005; 41 (3): 497-505.
Acute kidney disease Proc Natl Acad Sci US A. 2005; 102 (6): 2198-203
Furthermore, since activation of PXR promotes cholesterol degradation, the PXR activator of the present invention can be used as a therapeutic agent for metabolic diseases such as hypertension, arteriosclerosis, hyperlipidemia, obesity, and hypercholesterolemia. Useful (US Patent Application Publication No. 2004/0019027).

なお、一般式（I）の化合物がＰＸＲを活性化することは、例えば、後述の実施例に示すように、ＰＸＲ発現プラスミド、及びＰＸＲ結合配列とルシフェラーゼなどのレポーター遺伝子をつないだレポータープラスミドを用いたレポーターアッセイなどによって確認することができる。   The compound of the general formula (I) activates PXR using, for example, a PXR expression plasmid and a reporter plasmid in which a PXR binding sequence and a reporter gene such as luciferase are connected as shown in Examples below. It can be confirmed by a reporter assay or the like.

一般式（Ｉ）の化合物もしくはその塩又はそれらの溶媒和物とともに配合される「薬学的及び製剤学的に許容される担体」としては、賦形剤、崩壊剤、結合剤、滑沢剤、コーティング剤、色素、希釈剤、基剤、及び等張化剤などを用いることができる。
本発明のＰＸＲ活性化剤の剤型は特に制限されないが、経口投与の場合、錠剤、カプセル剤、散剤、細粒剤、顆粒剤、液剤、シロップ剤などを挙げることができ、非経口投与の場合、注射剤、点滴剤、及び坐剤などを挙げることができる。なお、本発明のＰＸＲ活性化剤を他剤と併用することもできる。 “Pharmaceutically and pharmaceutically acceptable carrier” formulated with the compound of general formula (I) or a salt thereof or a solvate thereof includes an excipient, a disintegrant, a binder, a lubricant, Coating agents, dyes, diluents, bases, tonicity agents and the like can be used.
The dosage form of the PXR activator of the present invention is not particularly limited. In the case of oral administration, tablets, capsules, powders, fine granules, granules, liquids, syrups, etc. can be mentioned. In some cases, injections, drops, suppositories and the like can be mentioned. In addition, the PXR activator of the present invention can be used in combination with other agents.

本発明のＰＸＲ活性化剤の投与経路は特に制限されず、経口的又は非経口的に投与することができる。投与量は年齢、体重、一般的健康状態、性別、食事、投与時間、投与方法、***速度、薬物の組合わせ、患者のそのときに治療を行っている病状の程度などに応じて適宜設定することができる。一日の投与量は患者の体重や状態、化合物の種類、投与経路などによって異なるが、例えば、非経口的には、皮下、静脈内、筋肉内、又は直腸内に約0.01〜50mg/人/日、好ましくは、0.01〜20mg/人/日投与され、経口的には、約0.01〜150mg/人/日、好ましくは、0.01〜100mg/人/日投与されることが望ましい。
The administration route of the PXR activator of the present invention is not particularly limited, and can be administered orally or parenterally. Dosage is appropriately set according to age, body weight, general health condition, sex, meal, administration time, administration method, excretion rate, combination of drugs, and the degree of medical condition being treated at the time of the patient. be able to. The daily dose varies depending on the weight and condition of the patient, the type of compound, the route of administration, etc., for example, parenterally, about 0.01-50 mg / person / person subcutaneously, intravenously, intramuscularly, or rectally. Preferably, it is administered at 0.01 to 20 mg / person / day, and orally, about 0.01 to 150 mg / person / day, preferably 0.01 to 100 mg / person / day.

以下、実施例を挙げて本発明を具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。なお、下記で試験化合物として用いた化合物は、特開平６−１３５９３８号公報に記載の方法に準じて製造されたものである。   Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples. The compounds used as test compounds below were produced according to the method described in JP-A-6-135938.

［実施例１］
ＰＸＲ活性化試験
ヒト肝癌由来細胞であるHepG2細胞において、ＰＸＲ発現プラスミド、及びＣＹＰ３Ａ4の発現調節領域にルシフェラーゼをつないだレポータープラスミドをトランスフェクトし、化合物のＰＸＲ活性化能を調べた。
ＰＸＲ発現プラスミドは、pTARGET Vector(Promega)にヒトＰＸＲ遺伝子（配列番号１）を挿入したものを用いた。レポータープラスミドは、pGL3-Basic Vecror（Promega）に、CYP3A4遺伝子の上流のXRE（xenobiotic responsive enhancer module：配列番号３および４）、dNR3（distal nuclear receptor binding element3：配列番号５）及び近位調節領域（proximal pregnane X receptor response element：配列番号６）を挿入したものを用いた（Drug Metabolism and Disposition vol. 32, No. 4, p468-472, 2004参照）。内部標準としてphRL-TK Vector（Promega）を用いた。
Minimum Essential Medium（GIBCO） 500mLにFBS（ウシ胎児血清）10％、ピルビン酸ナトリウム 1％、GlutaMAX （GIBCO）1％を加えた培地を用いてHepG2細胞を培養した。96ウェルプレートに細胞を4.5×104／ウェルの濃度でまき、24時間培養した。次いで、Lipofectamine 2000（Invitrogen）を用いて、PXR発現プラスミド、レポータープラスミド、及び内部標準プラスミドをトランスフェクションした。24時間後、各濃度の試験化合物（DMSO溶液）を添加し、さらに、24時間インキュベートした。インキュベート終了後、Dual-Glo Luciferase Assay System（Promega）を用いてルシフェラーゼ活性を測定した。
リファンピシン（シグマアルドリッチジャパン株式会社）及び化合物１についてレポータージーンアッセイを行った結果を表１及び図２に示した。リファンピシン、化合物１ともに濃度依存的にPXRを活性化し、EC50はそれぞれ、0.634μM（リファンピシン）、0.084μM（化合物１）であった。その結果、化合物１はリファンピシンよりも低濃度でＰＸＲを活性化することがわかった。 [Example 1]
PXR Activation Test In HepG2 cells, which are cells derived from human liver cancer, a PXR expression plasmid and a reporter plasmid in which the expression control region of CYP3A4 was linked to luciferase were transfected, and the ability of the compound to activate PXR was examined.
As the PXR expression plasmid, a plasmid obtained by inserting the human PXR gene (SEQ ID NO: 1) into pTARGET Vector (Promega) was used. The reporter plasmid is composed of pGL3-Basic Vecror (Promega), XRE upstream of the CYP3A4 gene (xenobiotic responsive enhancer module: SEQ ID NO: 3 and 4), dNR3 (distal nuclear receptor binding element3: SEQ ID NO: 5) and proximal regulatory region (SEQ ID NO: 5). An insertion of proximal pregnane X receptor response element (SEQ ID NO: 6) was used (see Drug Metabolism and Disposition vol. 32, No. 4, p468-472, 2004). PhRL-TK Vector (Promega) was used as an internal standard.
HepG2 cells were cultured in a medium containing 500% Minimum Essential Medium (GIBCO) plus 10% FBS (fetal bovine serum), 1% sodium pyruvate, and 1% GlutaMAX (GIBCO). Cells were seeded in a 96-well plate at a concentration of 4.5 × 10 4 / well and cultured for 24 hours. Subsequently, Lipofectamine 2000 (Invitrogen) was used to transfect the PXR expression plasmid, the reporter plasmid, and the internal standard plasmid. After 24 hours, each concentration of test compound (DMSO solution) was added and further incubated for 24 hours. After completion of the incubation, luciferase activity was measured using Dual-Glo Luciferase Assay System (Promega).
The results of reporter gene assay for rifampicin (Sigma Aldrich Japan Co., Ltd.) and Compound 1 are shown in Table 1 and FIG. Both rifampicin and compound 1 activated PXR in a concentration-dependent manner, and EC50s were 0.634 μM (rifampicin) and 0.084 μM (compound 1), respectively. As a result, it was found that Compound 1 activates PXR at a lower concentration than rifampicin.

Figure 2009120486
Figure 2009120486

その他の化合物についても、レポータージーンアッセイによりＰＸＲ活性化能を調べた。その結果、下記に示す化合物もＰＸＲを活性化することがわかった。

Figure 2009120486
Other compounds were also examined for their ability to activate PXR by reporter gene assay. As a result, it was found that the compounds shown below also activate PXR.
Figure 2009120486

［実施例２］
RT-PCRによるCYP3A4の発現量の解析
次に、化合物１及びリファンピシンが培養細胞においてＣＹＰ３Ａ４のｍＲＮＡ量を増
加させるかについて調べた。
HepG2細胞に0.25μＭ、１μＭ、もしくは４μＭの化合物１または１０μＭのリファンピシンを添加し、37℃で4日間培養した。培養終了後、トリゾール（Invitrogen社）を用いて細胞からトータルRNAを単離した。１μｇのトータルRNAを、ランダムヘキサマー及びSuperScriptII Transcription System（Invitrogen社）を用いて逆転写しｃＤＮＡを合成した。得られたｃＤＮＡを鋳型にして、５’−プライマー（配列番号７）、３’−プライマー（配列番号８）及び蛍光プローブ（配列番号９）を用いてリアルタイム定量ＰＣＲを行った。反応及び検出はPRISM7900 Sequence Detection System（Applied Biosystems）を用い、反応は５０℃、２分、９５℃、１０分の後に、９５℃、１５秒、６０℃、１分を４０サイクルの条件で行った。CYP3A4の発現レベルはβ-アクチンの発現量で標準化した。
結果を図２に示す。化合物１がリファンピシンと同等以上にＣＹＰ３Ａ４のｍＲＮＡ量を増加させる作用があることがわかった。 [Example 2]
Analysis of CYP3A4 expression level by RT-PCR Next, it was examined whether Compound 1 and rifampicin increase the amount of CYP3A4 mRNA in cultured cells.
HepG2 cells were added with 0.25 μM, 1 μM, or 4 μM Compound 1 or 10 μM rifampicin and cultured at 37 ° C. for 4 days. After completion of the culture, total RNA was isolated from the cells using Trizol (Invitrogen). 1 μg of total RNA was reverse transcribed using random hexamers and SuperScript II Transcription System (Invitrogen) to synthesize cDNA. Using the obtained cDNA as a template, real-time quantitative PCR was performed using a 5′-primer (SEQ ID NO: 7), 3′-primer (SEQ ID NO: 8) and a fluorescent probe (SEQ ID NO: 9). The reaction and detection were performed using PRISM7900 Sequence Detection System (Applied Biosystems), and the reaction was performed at 50 ° C., 2 minutes, 95 ° C., 10 minutes, 95 ° C., 15 seconds, 60 ° C., 1 minute under 40 cycles. . The expression level of CYP3A4 was normalized by the expression level of β-actin.
The results are shown in FIG. It was found that Compound 1 has an effect of increasing the amount of CYP3A4 mRNA at least as much as rifampicin.

本発明によれば、フタラジン誘導体の新規用途を提供することができる。   ADVANTAGE OF THE INVENTION According to this invention, the novel use of a phthalazine derivative can be provided.

リファンピシン（RIF）または化合物１を細胞に添加したときのCYP3A4のmRNA量を示す図。The figure which shows the amount of mRNA of CYP3A4 when rifampicin (RIF) or compound 1 is added to a cell. リファンピシン（RIF）または化合物１による濃度依存的なレポーター遺伝子の活性化を示す図。The figure which shows activation of a reporter gene depending on a density | concentration by rifampicin (RIF) or compound 1. FIG.

Claims (6)

下記一般式（I）で表される化合物もしくはその医薬的に許容される塩、又はそれらの溶媒和物を含むプレグナンＸ受容体活性化剤。
Figure 2009120486
 上記式中で、Ｒ1 はシクロヘキシル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいチエニル基；Ｃ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフリル基；チアゾリル基；フェノキシ基；Ｃ7〜Ｃ9のフェニルアルキル基；フェニルチオ基；モルフォリノ基；ピペリジル基；ピロリジニル基；ピリジル基；又はイミダゾリル基を表し、
Ｒ2 は−ＣＨＲ3 Ｒ4 （Ｒ3 は水素原子またはＣ1 〜Ｃ4 のアルキル基を表し、Ｒ4 はＣ1 〜Ｃ4 のアルキル基；シクロヘキシル基；チエニル基；またはＣ1 〜Ｃ4 のアルキル基、Ｃ1 〜Ｃ4 のアルコキシ基およびハロゲン原子から選ばれる１以上の置換基を有していてもよいフェニル基を表す。）；又はシクロヘキシル基を表し、
環Ａはベンゼン環、チオフェン環またはフラン環を表す。 A pregnane X receptor activator comprising a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof.
Figure 2009120486
 In the above formula, R 1 represents a cyclohexyl group; C 1 -C 4 alkyl groups, C 1 -C 4 one or more may have a substituent phenyl group selected from an alkoxy group and a halogen atom; C 1 -C 4 alkyl groups, C 1 -C 4 one or more thienyl group which may have a substituent selected from alkoxy groups and halogen atoms, alkyl groups of C 1 ~C 4, C 1 ~C A furyl group optionally having one or more substituents selected from 4 alkoxy groups and a halogen atom; a thiazolyl group; a phenoxy group; a C 7 to C 9 phenyl alkyl group; a phenylthio group; a morpholino group; a piperidyl group; Represents a pyrrolidinyl group; a pyridyl group; or an imidazolyl group,
R 2 represents —CHR 3 R 4 (R 3 represents a hydrogen atom or a C 1 to C 4 alkyl group, R 4 represents a C 1 to C 4 alkyl group; a cyclohexyl group; a thienyl group; or C 1 to C 4. alkyl group, optionally having one or more substituents selected from alkoxy groups and halogen atoms of C 1 -C 4 represents a phenyl group);. or cyclohexyl group,
Ring A represents a benzene ring, a thiophene ring or a furan ring. 請求項１に記載のプレグナンＸ受容体活性化剤を含むＣＹＰ３Ａ４誘導剤。 A CYP3A4 inducer comprising the pregnane X receptor activator according to claim 1. 請求項１に記載のプレグナンＸ受容体活性化剤を含む肝疾患治療薬。 The liver disease therapeutic agent containing the pregnane X receptor activator of Claim 1. 肝疾患が胆汁うっ滞性肝疾患又は黄疸である、請求項３に記載の肝疾患治療薬。 The therapeutic agent for liver disease according to claim 3, wherein the liver disease is cholestatic liver disease or jaundice. 請求項１に記載のプレグナンＸ受容体活性化剤を含む急性腎疾患治療薬。 A therapeutic agent for acute kidney disease comprising the pregnane X receptor activator according to claim 1. 請求項１に記載のプレグナンＸ受容体活性化剤を含む代謝性疾患治療薬。 A therapeutic agent for metabolic diseases comprising the pregnane X receptor activator according to claim 1.
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